The anthrax lethal factor (LF) is known to be pathogenically important, since anthrax bacilli lacking the LF gene show diminished virulence. Only a single cellular target for LF is known: the host macrophage, which undergoes apoptosis as a result of LF internalization. The killing of macrophages is also believed to be pathogenically important, because macrophage depletion paradoxically protects animals against B. anthracis infection. But the mechanism by which LF kills host macrophages has remained unclear. The events set in motion by LF have been analyzed using a combination of biochemical and genetic methods. It is known that LF is a zinc protease, and it has been shown to degrade MAP kinase kinases. Moreover, it is known that mutations of the gene encoding Kif1C, a kinesin-like cytoskeletally-associated protein, also render the host cell susceptible to LF action. Yet, to the present time, a clear sequence of events linking the LF protein to death of the macrophage has not emerged, and certainly, not all of the proteins that participate in LF toxicity have been identified. In order to find more of them, a forward genetic strategy is proposed. Germline mutations will be induced in mice using N-ethyl-N-nitrosourea (ENU), and macrophages isolated from the mutants will be systematically screened for resistance or hypersusceptibility to LF. Among 1638 F1 and 3153 F3 germline mutant mice analyzed to date, a single transmissible mutation that creates strong resistance to LF has been identified. This autosomal recessive mutation, Lfr1, appears to be required for normal signaling induced by activation of several of the mammalian TLRs, since TNF production in macrophages from Lfr1 homozygotes show defective signaling in response to lipopolysaccharide (LPS), bacterial lipopeptide, peptidoglycan, and unmethylated DNA bearing CpG motifs. The Lfr1 mutation will be mapped to a critical region and cloned. Moreover, we will extend our effort to approach saturation, and attempt to clone all mutations that show a strong phenotypic effect. The Lfr1 mutation, and all other mutations that are acquired through this forward genetic approach, will be subjected to detailed phenotypic analysis to determine the level at which they affect LF resistance or susceptibility. In this manner, it may be possible to infer a clear sequence of biochemical events linking LF internalization to the death of the cell.